miR-20a调控P-gp对非小细胞肺癌细胞A549顺铂化疗敏感性的影响

Effect of miR-20a-regulated P-gp on cisplatin chemosensitivity of non-small cell lung cancer cell line A549

目的探讨miR-20a调控P-gp对非小细胞肺癌细胞A549顺铂(DDP)化疗敏感性的影响。方法体外培养A549细胞并诱导产生A549/DDP细胞,RT-PCR方法检测miR-20a和P-gp mRNA表达情况。通过生物信息学网站预测miR-20a与P-gp是否有潜在的结合位点,通过荧光素酶报告基因实验进行验证。按Lipofectamine2000说明书方法分别将miR-NC和miR-20a mimic转染到A549/DDP细胞,检测不同浓度DDP对细胞增殖率的影响,确定半数抑制浓度,采用流式细胞术检测细胞凋亡,Western blot方法检测细胞中P-gp、Caspase-9和Caspase-3蛋白表达情况。结果A549/DDP细胞中miR-20a表达水平降低,P-gp mRNA表达水平增加(P<0.05),P-gp是miR-20a的靶点。随着DDP浓度增加,细胞增殖率降低(P<0.05);相同DDP浓度下,miR-20a mimic组细胞增殖率显著降低(P<0.05)。用DDP浓度为40.00μmol/L作为干预浓度进行后续检测,与miR-NC组相比,miR-20a mimic组细胞凋亡率、Caspase-9和Caspase-3蛋白表达增加,P-gp蛋白表达降低(P<0.05)。结论miR-20a通过靶向抑制P-gp促进A549/DDP细胞凋亡,增加耐药A549细胞DDP化疗敏感性。

Objective To investigate the effect of miR-20a-regulated P-gp on cisplatin(DDP)chemosensitivity of non-small cell lung cancer cell line A549.Methods A549 cells was culture in vitro and A549/DDP cells was induced.The expressions of miR-20a and P-gp mRNA were detected by RT-PCR.The bioinformatics website was used to predict whether there was a potential binding site between miR-20a and P-gp,which was verified by luciferase reporter gene experiment.The A549/DDP cells were transfected with miR-NC and miR-20a mimic respectively according to the Lipofectamine 2000 method.The effects of DDP at different concentrations on cell proliferation rate were detected,and the half inhibitory concentration was determined.The apoptosis of cells was detected by flow cytometry.The expressions of P-gp,Caspase-9 and Caspase-3 were detected by Western blot.Results The level of miR-20a in A549/DDP cells was decreased,while the level of P-gp mRNA was increased(P<0.05).P-gp was the target of miR-20a.With the increase of DDP concentration,the proliferation rate of cells was decreased(P<0.05).The proliferation rate of cells of miR-20a mimic group was significantly decreased under the same DDP concentration(P<0.05).DDP concentration of 40.00μmol/L was used as the intervention concentration for follow-up detection.Compared with miR-NC group,apoptosis rate,expressions of Caspase-9 and Caspase-3 were increased in miR-20a mimic group,while the expression of P-gp was decreased(P<0.05).Conclusion miR-20a promotes A549/DDP cell apoptosis through targeted inhibition of P-gp,and increases the sensitivity of drug-resistant A549 cells to DDP chemotherapy.